PepPlot shows several common measures
of protein secondary structure together
on one coordinated
plot. Most of the
curves are the average, sum,
or product of some residue-specific
attribute within a
window. In a few
cases, the attribute is both
specific to the residue and
dependent on its position in
the
window. Throughout the plot,
the blue curves are for
beta-sheets and the red curves
are for
alpha-helices; black is used for
turns and hydropathy. If
your plotter does not have
four colors, then
dashed lines are for alpha-helix
and solid lines are for
beta-structures.

This document is only a
description of what PepPlot does.
You may want to
read some of the articles
cited below to help you
interpret what the curves really
mean.

There are ten different panels
that can be plotted in
any combination and in any
order. In the
descriptions below they are referred
to from top to bottom
as if you had plotted
them all in the default
order as in the example
session and figure.

The Sequence

The first part of the
plot shows the sequence itself.
This panel is extremely
crowded if you use a
density of more than 100
residues per page.

The Residue Schematic

The second part of the
plot shows a schematic representation
of the sequence. Each
residue is
represented by a line at
the position where it occurs
in the sequence. The
lengths and colors of
the lines are used to
indicate chemically similar groups of
amino acids as follows.

The third panel is a
display of the residues that
are beta-sheet forming and breaking
as defined
by Chou and Fasman (Adv. Enz. 47;
45-147 (1978)). To nucleate
beta-structures, there should be
at least three beta-forming residues
and not more than one
breaking residue within a window
of
five.

Chou and Fasman Alpha and
Beta Propensities

The fourth panel of the
plot shows the Chou and
Fasman (1978 cited above) propensity
measures
for alpha-helix and beta-sheet.
As each curve rises past
the threshold for its color,
it satisfies one
criterion for propagation of an
alpha-helix or beta-sheet structure.
If the curves for alpha
and
beta propagation drop below the
black threshold (at value of
the 1.00 level) and if
there is at
least one breaking residue in
four, then the structure may
terminate. Both curves are
the
average of a residue-specific attribute
over a window of four.

Chou and Fasman Alpha-Helix Forming
and Breaking Residues

The fifth panel shows the
residues that are alpha-helix forming
and breaking, as defined by
Chou and Fasman (1978 cited
above). For alpha-helices to
nucleate, there should be four
or more
alpha-forming residues and not more
than one breaking residue within
six residues.

Chou and Fasman Amino Ends

The sixth panel shows regions
of the sequence that resemble
sequences typically found at the
amino end of alpha-helices and
beta-structures (Chou and Fasman, 1978
cited above). The
curves plot the probabilities for
a window of six that
the first three residues in
the window
precede the end of the
structure and the last three
residues are within the structure.
There are
two different residue-specific attributes used,
one for each half of
the product.

Chou and Fasman Carboxyl Ends

The seventh panel shows regions
of the sequence typically found
at the carboxyl end of
alpha-helices and beta-structures (Chou and
Fasman, 1978 cited above).
The two curves show
the probability for a window
of six that the first
three residues in the window
are within the
structure and the last three
residues are outside the structure.
Two different residue-specific
attributes are used, one for
each half of the product.

Chou and Fasman Turns

The eighth panel shows regions
of the sequence typically found
in turns (Chou and Fasman,
1978
cited above). The curve
is the product of a
residue-specific, position-dependent attribute
(probability) multiplied across a window
of four. The calculated
values are multiplied by 10,000
for plotting.

Hydrophobic Moment

The ninth panel shows the
helical hydrophobic moment at each
position of the sequence.
These
curves rise when the molecule
forms either an alpha-helix or
a beta-sheet at the interface
between the solvent and the
interior of the molecule.
Said another way, the moment
statistic is
the probability that the sequence
at each position is amphiphilic,
that is, it appears to
have
hydrophobic residues on one side
and hydrophilic residues on the
other. The hydrophobic
moment is calculated as described
by Eisenberg et al.
(Proc. Natl. Acad. Sci. USA 81; 140-144
(1984)), except that we have
normalized the hydrophobic moment for
the local hydrophobicity of
the amino acids in the
window where the moment is
being determined. This makes
the method
equivalent to that described by
Finer-Moore and Stroud (Proc. Natl. Acad. Sci. USA, 81;
155-159
(1984)).

In a typical alpha-helix, each
residue is oriented about 100
degrees from the preceding residue.
The alpha moment that we
plot in this panel is
the maximum for all inter-residue
angles
between 95 and 105 degrees
The alpha moment curve is
calculated for a window of
eight
residues.

Typical beta-strands have 160 degrees
of rotation between adjacent residues.
The beta
hydrophobic moment curve is the
maximum for all inter-residue angles
between 159 to 161
degrees calculated over a window
of six residues.

Kyte and Doolittle Hydropathy

The tenth panel has two
curves based on the average
hydrophobicity. The black curve
is the
Kyte and Doolittle hydropathy measure
(J. Mol. Biol. 157; 105-132 (1982)). This
curve is the
average of a residue-specific hydrophobicity
index over a window of
nine residues. When the
line
is in the upper half
of the frame, it indicates
a hydrophobic region, and when
it is in the lower
half, a hydrophilic region.
You can set the Kyte-Doolittle
window to a number other
than nine
using -HWINdow=n.

Goldman, Engelman, and Steitz Transbilayer
Helices

The green curve in the
tenth panel is the Goldman,
Engelman, and Steitz (GES) curve
for
identifying nonpolar transbilayer helices (reviewed
in Ann. Rev. Biophys. Biophys. Chem. 15;
321-353 (1986)). The curve
is the average of a
residue-specific hydrophobicity scale (the GES
scale) over a window of
20 residues. When the
line is in the upper
half of the frame, it
indicates
a hydrophobic region and when
it is in the lower
half, a hydrophilic region.
You can suppress the
GES curve in this panel
with -NOGES. You can
set the GES window to
a number other than 20
with -GESWindow=n.

Garnier Predictions Can Be Written
Into a File

Using -GARnier, secondary structure prediction
using the method of Garnier,
et
al. (J. Mol. Biol. 120; 97-120 (1978)) can also
be calculated by PepPlot and
written into a file.

Here is a session using
PepPlot to plot the secondary
structure measures for the first
100 residues of
adenylate kinase (PIR:Kihua). This
session with PepPlot also writes
the Garnier predictions, Chou and
Fasman values, and helical hydrophobic
moment values to separate output
files.

Secondary structure prediction using the
method of Garnier et al. (J. Mol. Biol. 120;
97-120 (1978)) is
also performed by PepPlot when
the program is run with
-GARnier. The Garnier method
calculates a
statistic for alpha-helix, beta-sheet, turns,
and random coil structures using
position-dependent,
residue-specific information within a window
of 17. The structure
predicted for the residue in
the
center of the window is
the structure with the largest
calculated statistic at that position.

Output File Structure

The results of the Garnier
prediction are written into a
file. The file shows
different predictions
for several different combinations of
decision constants (see below).
The predicted structure is
represented with an A for
alpha, B for beta, C
for random coil, and T
for turn. Question marks
indicate that two or more
structures are equally probable.

Decision Constants From Physical Measurements

If you have physical data
on the proportion of the
protein's secondary structure that is
alpha-helix and beta-strand, decision constants
("fudge factors") can be used
to bias the Garnier
predictions. Read predictions from
that column of data whose
percent alpha-helix and percent
beta-strand corresponds most closely to
the physical measurements for the
entire protein.

Decision Constants Without Physical Measurements

If you have no physical
measurement of the percentage of
alpha-helix and beta-strand in your
protein, Garnier recommends using the
percent alpha and beta with
no decision constants (the
No DC column of data
in the file).

With -CFFile, PepPlot writes a
file with the Chou and
Fasman (1978, cited above) values
for every
position in the sequence written
out as a table of
numbers. Here is part
of the Chou and Fasman
output file, kihua.cho:

With -MOMentfile, PepPlot writes a
file with the helical hydrophobic
moment values for every
position in the sequence written
out as a table of
numbers. Here is part
of the moment output file,
kihua.mom:

PeptideStructure and PlotStructure were sent
to us by Dr. Berthold Foertsch
of the Max Planck
Institute of Munich. Used
together, these two programs let
you see a graphics representation
of the
best choice Chou-Fasman or Garnier
prediction with hydrophobicity or antigenic
index superimposed.

Moment makes a contour plot
of the helical hydrophobic moment
for all rotation angles between
0 and
180 degrees per residue (Eisenberg,
1984, and Finer-Moore and Stroud,
1984, cited above).
HelicalWheel plots a peptide sequence
as a helical wheel to
help you recognize amphiphilic regions.

The residue-specific attributes for all
of the measurements in PepPlot
are only defined for the
standard
alphabet of protein sequence characters,
including B, X, Z, and
* (see Appendix III). Sequences
containing any other symbols, such
as the gap symbols period
(.) and tilde (~), are
not suitable as input
for PepPlot.

You should realize that secondary
structure predictions are not very
reliable, especially for proteins
that are not soluble or
globular.

Plots with more than about
250 residues per 100 platen
units may be too compressed
to be useful for
structure prediction, although they may
be useful for comparing two
protein sequences for structurally
similar regions. When multiple-page
plots are made, successive pages
are overlapped by one residue
so
that the plots can be
spliced together. The curves
stop one-half window width from
the ends of the
sequence.

The Wisconsin Package must be
configured for graphics before you
run any program with graphics
output!
If the % setplot
command is available in your
installation, this is the easiest
way to
establish your graphics configuration, but
you can also use commands
like % postscript that
correspond to the graphics languages
the Wisconsin Package supports.
See Chapter 5, Using Graphics
in the User's Guide for
more information about configuring your
process for graphics.

If you need to stop
this program, use <Ctrl>C to
reset your terminal and session
as gracefully as
possible. Searches and comparisons
write out the results from
the part of the search
that is complete
when you use <Ctrl>C.
The graphics device should stop
plotting the current page and
start
plotting the next page.
If the current page is
the last page, plotters should
put the pen away and
graphic terminals should return to
interactive mode.

PepPlot uses dashed lines when
four-color plotting is not available.
Alpha curves are red
when color is
available, dashed in black and
white. Beta curves are
blue in color, solid in
black in white. In
the
hydrophilicity panel, the GES curve
is green if color is
available and dashed otherwise.
In the residue
schematic, hydrophilic and charged residues
are red and green in
the color plot and dashed
in black
and white. Hydrophobic residues
are blue in color and
solid in black and white.

There are three threshold lines
across the Chou-Fasman panel (panel
D). From top to
bottom, these
lines are as follows: the
blue line is the threshold
for the beginning of a
beta-sheet; the red line is
the
threshold for the beginning of
an alpha-helix; and the black
line is the breaking line
below which either
kind of structure is no
longer predicted. In black
and white these lines are
solid, short dashed, and long
dashed, respectively.

All parameters for this program
may be added to the
command line. Use -CHEck
to view the summary
below and to specify parameters
before the program executes.
In the summary below, the
capitalized
letters in the parameter names
are the letters that you
must type in order to
use the parameter.
Square brackets ([ and ])
enclose parameter values that are
optional. For more information,
see "Using
Program Parameters" in Chapter 3,
Using Programs in the User's
Guide.

PepPlot was written by Drs. Michael
Gribskov and John Devereux of
the Genetics Computer Group.
It
was first described in Nucl. Acids
Res. 14(1); 327-334 (1986). The
original code was revised by
John
Devereux to support command-line control
for Version 5 and to
support plotting the panels
independently for Version 6.

The files described below supply
auxiliary data to this program.
The program automatically reads
them from a public data
directory unless you either 1)
have a data file with
exactly the same name in
your current working directory; or
2) name a file on
the command line with an
expression like
-DATa1=myfile.dat. For more information
see Chapter 4, Using Data
Files in the User's Guide.

PepPlot reads three different data
files to find the residue-specific
attributes: pepplot.dat, which
contains Chou-Fasman and hydropathy values;
ges.dat, which contains the GES
scale; and garnier.dat,
which contains the Garnier measures.

You can set the parameters
listed below from the command
line. For more information,
see "Using
Program Parameters" in Chapter 3,
Using Programs in the User's
Guide.

-DENsity=87

sets the number of bases
or amino acids per 100
platen units (PU). This
is usually equivalent to
the number of bases or
amino acids per page.
Output from different GCG graphics
programs
that are run at the
same density can be compared
by lining up the plots
on a light box.

causes PepPlot to write an
output file with the Chou
and Fasman predictions for the
sequence.
The filename is the sequence
name plus the filename extension
.cho, unless you set it
to
something else.

-GARnierfile=kihua.gar

causes PepPlot to write an
output file with the Garnier
predictions for the sequence.
The
filename is the sequence name
plus the filename extension .gar,
unless you set it to
something
else.

-MOMentfile=kihua.mom

causes PepPlot to write an
output file with the helical
hydrophobic moments for the sequence.
The filename is the sequence
name plus the filename extension
.mom, unless you set it
to
something else.

-HWINdow=9

sets the window size for
calculating the Kyte and Doolittle
hydropathy curve. The hydropathy
window size must be between
1 and 50.

-GESWindow=20

sets the window size for
calculating the Goldman, Engelman, and
Steitz hydropathy curve. The
GES window size must be
between 1 and 50.

-NOGES

suppresses the Goldman, Engelman, and
Steitz curve in the hydropathy
(eighth) panel.

-SHOwseq

The sequence display in the
top panel is normally suppressed
if it seems too crowded.
Use this
parameter to insist that it
be plotted no matter how
crowded it seems.

-BOXES

draws a box around each
quantitative panel (the ones with
the tick marks).

-NOTITle

suppresses the plot's title.

-NOPLOt

suppresses the plot.

The parameters below apply to
all Wisconsin Package graphics programs.
These and many others
are
described in detail in Chapter 5, Using Graphics
of the
User's Guide.

-FIGure=programname.figure

writes the plot as a
text file of plotting instructions
suitable for input to the
Figure program
instead of sending it to
the device specified in your
graphics configuration.

-FONT=3

draws all text characters on
the plot using Font 3
(see Appendix I).

-COLor=1

draws the entire plot with
the pen in stall 1.

The parameters below let you
expand or reduce the plot
(zoom), move it in either
direction (pan), or
rotate it 90 degrees (rotate).

-SCAle=1.2

expands the plot by 20
percent by resetting the scaling
factor (normally 1.0) to 1.2
(zoom in). You
can expand the axes independently
with -XSCAle and -YSCAle.
Numbers less than 1.0
contract the plot (zoom out).

-XPAN=30.0

moves the plot to the
right by 30 platen units
(pan right).

-YPAN=30.0

moves the plot up by
30 platen units (pan up).

-PORtrait

rotates the plot 90 degrees.
Usually, plots are displayed
with the horizontal axis longer
than the
vertical (landscape). Note that
plots are reduced or enlarged,
depending on the platen size,
to fill
the page.

-NOCLIpping

If the data points on
a line fall outside of
the window in which the
data are supposed to be
represented, most programs will clip
the graph at the edge
of the window. This
switch disables
that clipping.

Licenses and Trademarks Wisconsin
Package is a trademark of Genetics Computer Group, Inc. GCG and the
GCG logo are registered trademarks of Genetics Computer Group,
Inc.

All other product names mentioned in this documentation may
be trademarks, and if so, are trademarks or registered trademarks of
their respective holders and are used in this documentation for
identification purposes only.